CN1523354A - Method and apparatus for bio-molecular chip minute quantity sample application and reaction - Google Patents
Method and apparatus for bio-molecular chip minute quantity sample application and reaction Download PDFInfo
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- CN1523354A CN1523354A CNA031026591A CN03102659A CN1523354A CN 1523354 A CN1523354 A CN 1523354A CN A031026591 A CNA031026591 A CN A031026591A CN 03102659 A CN03102659 A CN 03102659A CN 1523354 A CN1523354 A CN 1523354A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/02—Burettes; Pipettes
- B01L3/0289—Apparatus for withdrawing or distributing predetermined quantities of fluid
- B01L3/0293—Apparatus for withdrawing or distributing predetermined quantities of fluid for liquids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0819—Microarrays; Biochips
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
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Abstract
The invention relates to a method to directly add trace sample and instantly react on the biomolecule chip and device. The method includes: under the action of external pressure, the surfaces of microarray and smooth chips close contact to form a sealed cavity. The bio-ligand molecule which needs fixing enters the cavity through the micro-flow channel and reacts with the chip surfaces, and after reacting, the ligand molecule is discharged through the micro-flow channel. The device includes: elastic material slice carved with grooves, microarray arranged of grooves and micro-flow channel matching the microarray; and elastic material membrane covered on the micro-flow channel, where there is a liquid entrance made on the base slice of microarray or micro-flow channel. After fixed and reacting, the chip surfaces are washed by buffer liquor, to make fixation and reaction of the biomolecule on the surface uniform and consistent, effectively heightening the detecting quality.
Description
Technical field
The present invention relates to a kind of biomolecule chip production method and device thereof, relate in particular to a kind of device that directly on the biomolecule chip, carries out the method for micro sample-adding and immediate reaction and realize this method.
Background technology
The biomolecule chip is a kind of integrated parallel Measurement for Biotechnique that just grew up in recent years, can integrated multiple aglucon on small geometric scale, so just can be simultaneously the multiple index of micro-example be detected.Because biomolecule sample price height requires consumption the least possible, therefore require employed biomolecule reagent of biomolecule chip and sample traceization, this also just requires chip application of sample and reaction unit microminiaturization.What at present, the application of sample of biochip mainly adopted is point sample instrument.Difference according to the point sample mode is divided into two classes, and a class is a contact, at first dip in and get stand-by aglucon with spotting needle, then by the contact chip surface aglucon point on chip; One class is the spray printing formula, draws a spot of aglucon for the treatment of a little with hollow spotting needle earlier, and the mode by similar ink-jet printer is added to aglucon on the chip surface then.The common drawback of this dual mode is that the point sample amount is inhomogeneous, and the surface density of the interior aglucon molecule of a single point distributes also inhomogeneous, and this will have a strong impact on the quality of testing result.Current, what biochip reaction adopted is the W-response mode mostly, exactly whole being immersed in the testing sample solution of chip is reacted.The testing sample solution amount that this method needs is more, and the reaction time is long, and sensitivity is not high.
Another chip application of sample and reaction technology are that fluid channel transports and the microcavity reactor.At present, generally the chip of the fluid channel technology of Shi Yonging is incorporate, be that chip and fluid channel are to be produced on the same block of material, as document 1:Dielectrophoretic cell separation and gene expression profiling onmicroelectronic chip arrays.July 15,2002 Huang Y, Joo S, Duhon M, Heller M, Wallace B, Xu X Anal Chem 2002 Jul 15; 74 (14): described among the 3362-71.The employed fluid channel of this method is disposable use, and this fluid channel is made complicated, and cost is higher, makes to use to be restricted.Carry out biomolecule operation fixing and detection reaction simultaneously and divide in two kinds of devices and carry out for 2 times, therefore, preparation technology is loaded down with trivial details.
Summary of the invention
The objective of the invention is in order to overcome the shortcoming of above-mentioned prior art; For the cost of manufacture that reduces biochip significantly with simplify the technology of the fixing and detection reaction of biomolecule, thereby provide a kind of biochip to make and directly carry out on this biochip that biomolecule is fixed and the method and apparatus of detection reaction immediately.
The object of the present invention is achieved like this:
Biochip preparation method provided by the invention comprises that order is carried out as follows:
(1) base material with chip closely contacts with the microarray template, and compresses fluid channel by external force;
(2) the fine pipe of aglucon molecule by fluid channel is transported to the lip-deep selection area of chip base; By the time after the aglucon molecule is fixed on the chip base;
(3) chip base that is fixed with biomolecule that step (2) is prepared is washed with damping fluid, damping fluid is transported in the zones of different of chip surface by fluid channel; Wash the aglucon molecule that is not fixed on the chip surface;
(4) by a flexible sheet, the zone that is fixed with the aglucon molecule is together in series; Perhaps the switch that forms by the node on the extruding flexible sheet is together in series the zone that is fixed with the aglucon molecule;
(5) biological sample to be detected is transported in each unit on the chip surface that step (4) prepares by fluid channel again, carries out detection reaction immediately;
(6) take off step (5) preparation and the chip that is loaded with the detection reaction result that obtains, use chip detector to detect its reaction result.
Employed aglucon molecular conecentration is 0.001-1mg/ml in the described step (2); The flow velocity of described aglucon molecule in fluid channel is the 0.1-100 mul/min.
Biological sample is the 1-100 mul/min by the flow velocity of fluid channel in the described step (5).
Described chip base material comprises: silicon, glass, metal, plastic or other material or above-mentioned several compound substance, preferred silicon.
This method is combined fluid channel and microarray.Microarray is meant the micro-recesses that is produced on the rubber-like solid material, and it is fixed that the number of microarray further groove comes according to the index that detects, individual or more to hundreds of from one.Fluid channel is meant the pipeline of the small internal diameter that is connected with micro-recesses.
Microarray can closely contact the cavity that formation seals one by one with smooth chip surface under the effect of external pressure.Need fixing biological aglucon molecule to enter cavity and chip surface reacts by fluid channel, reacted aglucon molecule is discharged by fluid channel again.So just realized carrying out in the zones of different on chip surface micro sample-adding.Then, biological sample to be detected also is to enter each cavity by fluid channel, after the biological aglucon molecular reaction of having fixed on the chip surface, discharges by outlet again.Biochip is used for detecting simultaneously various biomolecules in the biological sample to be measured.Designed the FLOW CONTROL part in this method, make with a biological sample to be measured successively with chip on the multiple biological aglucon molecular reaction that pre-fixes, reduced amount of samples effectively.Can realize connecting between the arbitrary number groove by the FLOW CONTROL part.The fixing zone of aglucon molecule is that strictness limits on the chip, and fixing and reacted surface is washed through damping fluid again, can make the surface go up the fixing of biomolecule and reaction uniformity, has improved the quality of detection effectively.The chip reaction is limited in the tiny area, and under flow state, has quickened the mass transfer rate of biomolecule, has shortened the reaction time effectively, has improved sensitivity.Fixing and the reacted chip of biomolecule is with after the microarray template is separated, use chip detector detection reaction result.Can reuse with the microarray template after chip material separates, repeatedly to carry out the fixing and reaction of biomolecule on the chip.
The isolated plant of biochip preparation method provided by the invention is according to the mode difference that constitutes fluid channel, and this device comprises: two kinds.
At first narrate first kind, this device comprises: be carved with the elastic material sheet 3 of groove 2 on surface, its groove 2 is arranged by the array formula, and groove 2 two ends have first through hole 10; Wherein the through hole of each groove comprises two fluid channel of one-in-and-one-out; Also be carved with groove 2 ' on one surface of a rigid solid material block 4, the two ends of groove 2 ' have second through hole 11, and the groove one side that is carved with of elastic material sheet 3 is not with groove one side relative fixed together with rigid solid material block 4; Through hole on this rigid solid material block 4 and through hole on the elastic material sheet 3 corresponding and hole of staggering communicates, promptly first groove 2 through hole communicates as solution inlet port with a through hole of groove 2 ' as taphole, and the outlet of groove 2 ' communicates with the import of next groove 2; Solid pieces of material 4 two sides have a testing liquid import 7 and a testing liquid outlet 8, and a switch is installed in the import; Chip 1 closely contact on elastic material sheet 3, the pipe 12 that the plug-in mounting diameter is close in the through hole of solid pieces of material 4, fluid channel is with the formation that is connected of the through hole on the microarray template by pipe 12.
Groove on microarray template under the effect of external pressure closely contacts the cavity that formation seals one by one with smooth chip surface.Need the fixing aglucon molecule can be under the driving of pumping, enter cavity and chip surface reacts by fluid channel, reacted sample be discharged by fluid channel again.So just realized on the zones of different on the chip, carrying out micro sample-adding.Chip behind the application of sample needn't take off from device, can directly carry out detection reaction.Take off fine pipe from microarray, use elastic membrane closed pockets 2 ', groove 2 is together in series, stay next import and an outlet.Biological sample to be detected enters each cavity successively by import, and discharges by outlet after the aglucon molecular reaction fixing on the chip surface again.Reacted chip can take off from device, detects by the reaction result of detecting device to aglucon on the chip and acceptor.
Also comprise an elastic membrane and have an import and an outlet, perhaps have an import, have an outlet in elastic material block one side again in solid pieces of material one side in the solid pieces of material two sides; One switch is installed in its import; Chip behind the application of sample needn't take off from device, can directly carry out detection reaction.Take off fine pipe from microarray, use an elasticity membrane cover on solid pieces of material, closed pockets 2, groove on the elastic material sheet is together in series, relative with solution inlet port opens a hole at the elastic membrane place, as the hole of detecting the liquid import and the solid pieces of material side being opened as detecting taphole.Biological sample to be detected enters each cavity successively by solution inlet port, discharges by taphole after reacting with the biological sample of having fixed on the chip surface again.Reacted chip can take off from device, by detecting device the result is detected.
Direct preparation biochip of the present invention and second kind of structure of carrying out the device of detection reaction immediately comprise: fluid channel 5 also comprises the microarray template that is complementary with it; Wherein the microarray template comprises: be carved with the elastic material sheet 3 of at least 2 grooves 2 on the surface, and the facing up of elastic material sheet 3 fluted 2, a through hole 10 is opened at the two ends of each groove 2 respectively, and the another side of elastic material sheet 3 closely contacts with chip 1 surface; Fluid channel comprises: a resilient material diaphragm 13, on resilient material diaphragm 13, be shaped on chase 14, and produce another port 6 and extraneous UNICOM on through hole 10 positions of the corresponding microarray upper groove 2 of an end of its chase 14; Through hole 10 corresponding chases 14 of each groove; And the film of corresponding microarray resilient material diaphragm 13 through holes on the resilient material diaphragm 13 is got through pore-forming, on be with chase 14 resilient material diaphragms 13 again, cover one deck elastic material membrane again, and the two membranes periphery is bonded together, formed fluid channel 5 by chase 14, interconnect between the fluid channel 5, at node location, the method for pushing by external force forms first, second switch 15,17.
Also comprise a rigid solid material block 4, this solid pieces of material 4 is fixed on the face of resilient material diaphragm 3 band grooves 2, the through hole 10 that open at the two ends of elastic material membrane piece 3 grooves 2 runs through rigid solid material block 4, and resilient material diaphragm 16 does not have the elastic material membrane of chase and solid pieces of material 4 upper surfaces of microarray are bonded together.
Groove on microarray under the effect of external pressure can closely contact the cavity that formation seals one by one with smooth chip surface.During application of sample, push the pathway closure that makes between fluid channel, just close second switch by external force.Each groove part has and independently passes in and out runner.Under the driving of pumping, different aglucon protein solutions enters into cavity and chip surface reaction, thereby fixes from the teeth outwards.Use buffer solution for cleaning then, remove and be not fixed on proteins on surfaces.So just realized carrying out in the zones of different on chip the purpose of micro sample-adding.When carrying out detection reaction, open the second switch between fluid channel, close first switch on the fluid channel, so just a plurality of cavitys are cascaded, only stay next import, an outlet.Protein solution to be measured enters each cavity successively and is fixed on lip-deep aglucon proteins react along import; After the reaction, use buffer solution for cleaning, silicon chip takes off detection from device.
Described elastic material sheet comprises: silica gel, rubber, plastics, glass or other rubber-like material.
Described solid pieces of material comprises: materials such as silica gel, rubber, plastics, metal, glass, its thickness 1mm is to 10mm.
Described groove is a strip groove, and its strip groove area is from 0.01mm
2-1mm
2The degree of depth is from 10 μ m-1mm; At least 2 of the numbers of its strip groove for example can be from 2-1000.
Described through hole internal diameter can be from 10 μ m to 1mm.
The internal diameter of described fluid channel can be from 10 μ m to 1mm.
The internal diameter of described chase can be from 10 μ m to 1mm.
The invention has the advantages that:
Because the method that is used for directly preparing the biomolecule chip and carrying out detection reaction immediately of the present invention can and be carried out detection reaction immediately and carry out at same device the preparation biochip, and the fixing zone of aglucon biomolecule is strict the qualification on this chip, fixing and reacted surface is washed through damping fluid again, can make the surface go up the fixing of biomolecule and reaction uniformity, improve the quality that detects effectively.The chip reaction is limited in the tiny area, and under flow state, has quickened the mass transfer rate of biomolecule, has shortened the reaction time effectively, has improved sensitivity.Can reuse with the microarray template after the chip of making separates, carry out the fixing and reaction of biomolecule on the chip.
Description of drawings
Fig. 1 a is the microarray stencil plane synoptic diagram in first kind of device of the present invention
Fig. 1 b is the microarray template side view in first kind of device of the present invention
Fig. 2 is the structural drawing of first kind of device of the present invention when being used for directly preparing the biomolecule chip
(through hole at microarray template groove two ends and the through hole at rigid solid material block groove two ends stagger corresponding communicating form fluid channel and cooperate synoptic diagram)
Fig. 3 is the structure drawing of device of first kind of device of the present invention when being used for carrying out detection reaction immediately
Fig. 4 a is microarray (elastic material sheet) the stencil plane synoptic diagram in second kind of device of the present invention
Fig. 4 b is the microarray template side view of Fig. 4 a
Fig. 5 is the structural representation of second kind of device of the present invention
Fig. 6 is second kind of another kind of example structure synoptic diagram of device of the present invention
Fig. 7 is the fluid channel vertical view in second kind of device of the present invention
Drawing is described as follows:
1-chip 2 first groove 3-elastic material block or diaphragms
4-solid pieces of material 5-fluid channel 6-liquid entrance
7-testing liquid import 8-testing liquid outlet 9-sealing elastic membrane
The 10-first through hole 11-second through hole (not shown in FIG.) 2 '-the second groove
The flexible sheet 14-chase of 12-pipe 13-band chase
The 15-first switch 16-elastic material membrane (not shown in FIG.) 17-second switch
Embodiment
Below in conjunction with drawings and Examples the present invention is described in detail
Make first kind device, and the method for carrying out the fixing of 12 kinds of protein thereon and detecting.This device comprises 12 grooves.The groove area is 1mm
2, be 0.1mm deeply, fluid channel internal diameter 0.5mm.Resilient material is a rubber, and solid material is an organic glass, and pipe is a teflon, and the elastic material membrane that is used to seal is a silica gel.Chip material is a silicon.
Make a microarray template by Fig. 1-2; Employing thickness is that the rubber of 1mm is done elastic material block 3, and its above-listed configuration is arranged with 12 grooves 2, and these groove 2 areas are 1mm
2, be 0.1mm deeply; First through hole 10 of an internal diameter 0.5mm is opened as fluid channel 5 (as shown in Figure 1) in these groove 2 two ends.Employing thickness is that the organic glass of 5mm is done solid pieces of material 4, is arranged with 12 grooves 2 ' at plexiglass block 4 above-listed configurations, and this groove 2 ' area is 1mm
2, be 0.1mm deeply; Second through hole 11 of an internal diameter 0.5mm is opened at these groove 2 ' two ends; Wherein the through hole of every groove comprises two fluid channel 5 of one-in-and-one-out; The face of this sheet rubber 3 band grooves and plexiglass block 4 are not with the face relative fixed of groove not together; Second through hole 11 on this plexiglass block 4 communicates with first through hole 10 on the sheet rubber 3, and first through hole 10 at sheet rubber 3 lip-deep grooves 2 two ends staggers corresponding with the through hole 11 at plexiglass block 4 grooves 2 ' two ends, be that in second through hole 11 of groove 2 ' one first through hole 10 as taphole and groove 2 communicates as solution inlet port, the outlet of groove 2 ' communicates with the import of groove 1; Plexiglass block 4 two sides have 2 holes, and the import 7 of liquid is detected in one of them hole when detecting, and this import 7 installs a switch, and another hole is as the outlet 8 that detects liquid; Chip 1 closely contact is equipped with fine pipe 12 in second through hole 11 of plexiglass block 4 on sheet rubber 3, fluid channel 5 is with the formation (as shown in Figure 2) that is connected of second through hole 11 on the plexiglass block 4 by fine pipe 12.The pipe 12 of fluid channel 5 usefulness of present embodiment is a teflon, and the elastic material membrane 9 that is used to seal is silica gel.Chip 1 material is a silicon.
Use method of the present invention, carry out the fixing and detection of 12 kinds of protein in the device of the foregoing description, its step is as follows:
Under the effect of external pressure (as shown in Figure 2), silicon chip 1 closely contacts with microarray when (1) working, and forms 12 independently cavitys of sealing like this between silicon chip 1 and the groove 2, and each cavity has two fluid channel 5 of one-in-and-one-out;
(2) under the promotion of micro-ram pump, for example with 12 kinds of protein solutions, perhaps hepatitis B surface antigen, hepatitis B virus e antigen, hepatitis B virus core antigen, hepatitis B surface antibody, hepatitis B e antibody solution 10 microlitres (concentration is 0.1mg/ml) are transported to zones of different on the silicon chip by fluid channel respectively, flow speed control is in 1 mul/min, waits after the aglucon molecule is fixed on the chip 1;
(3) then, carry phosphate buffer to chip 1 surface of above-mentioned steps (2) preparation, wash the biomolecule that is not fixed on the silicon chip surface by fluid channel;
(4) take off the pipe 12 of fluid channel 5 usefulness, use the pellosil 9 of a sealing to cover on fluid channel 5, the zone that is fixed with the aglucon molecule is together in series;
(5) by injecting 100 microlitres patients serum to be checked to testing liquid import 7, be after 10 mul/min are injected reaction by a fluid channel, to flow out with flow velocity through testing liquid outlet 8, re-use phosphate buffer flushing (as shown in Figure 3);
(6) take off chip 1, use detecting device detection reaction result.
Enter cavity and silicon chip surface contact reaction by fluid channel, thereby proteinaceous solid is fixed on the silicon chip surface.Re-use buffer solution for cleaning silicon chip surface, cavity and fluid channel, the protein that is not fixed on the silicon chip surface is discharged.
Preparation one has 100 independently devices of the present invention of the cavity of sealing.
The elastic material block 3 of present embodiment is a silica gel, comprises on it being carved with 100 grooves 2.These groove 2 areas are 0.1mm
2Or 0.01mm
2Dark is 0.1mm; It is that 10, blocks of solid materials of first through hole 4 of 0.2mm are aluminium that groove 2 two ends have internal diameter, comprises on it and is carved with 100 grooves 2 '.This groove 2 ' area is 0.1mm
2, be 0.1mm deeply; Groove 2 ' two ends have second through hole 11 that internal diameter is 0.2mm.3 fluted of elastic material blocks and solid material 4 do not have the groove surface relative fixed, wherein first through hole 10,11 dislocation of second through hole communicate, form fluid channel 5, its fluid channel 5 internal diameter 0.2mm, the stainless steel pipe 12 that is inserted with suitable for reading of fluid channel 5, the suitable for reading of stainless steel pipe 12 is liquid-inlet 6, and all the other structures are with embodiment 1.
Comprise also that when detecting one is used to seal the pellosil 9 of usefulness, this pellosil 9 covers on the groove 2 ' of organic glass 4, and gold-plated glass material is as chip 1, and all the other structures are with embodiment 1.
Use the device of present embodiment, carry out the fixing and detection of 1000 kinds of genes, its step is as follows:
(1) when being prepared genetic chip, under the effect of external pressure, silicon chip closely contacts with the microarray template, has just formed 1000 independently cavitys of sealing like this between silicon chip and the groove, and each cavity has two fluid channel of one-in-and-one-out;
(2) under the promotion of micro-ram pump, respectively 1000 kinds of not homotactic dna molecular solution 10 microlitres (concentration is 0.1 μ g/ml) are transported to zones of different on the silicon chip by fluid channel, flow speed control is in 1 mul/min, wait dna molecular fixing after;
(3) then, use the phosphate buffer flushing, wash the molecule that is not fixed on the silicon chip surface;
(4) take off the pipe 12 of fluid channel 5 usefulness, use the pellosil 9 of a sealing to cover on fluid channel 5,1000 grooves are together in series, and the zone that is fixed with dna molecular is together in series;
(5) by inject the to be measured DNA sample of 100 microlitres to testing liquid import 7, be that 10 mul/min are injected, on a fluid channel is flowed through the chip that step (4) makes, after each zone reaction, flow out from testing liquid outlet 8 with flow velocity through degenerative treatments; The flushing of use phosphate buffer;
(7) take off silicon chip, use detecting device detection reaction result.
When detecting: the chip behind the above-mentioned application of sample needn't take off from device, can directly carry out detection reaction.On the microarray template, take off fine pipe 12, use the groove 2 ' on a pellosil 9 sealing organic glass 4,100 grooves on the elastic material sheet 3 are together in series, relative with solution inlet port opens a hole at the elastic membrane place, as detecting liquid import 7, with the hole that the solid pieces of material side is opened as detecting taphole 8, biological sample to be detected enters each cavity successively by solution inlet port 7, and discharges by taphole 8 after the biological sample reaction fixing on chip 1 surface again.Reacted chip 1 can take off from device, by detecting device the result is detected.
Make first kind device, as Figure 1-3; Adopt method of the present invention to carry out the fixing and detection of 100 kinds of protein.
This device comprises 100 grooves.The groove area is 0.06mm
2, be 0.1mm deeply, fluid channel internal diameter 0.2mm.Resilient material is a silica gel, and solid material is an aluminium, and pipe is a stainless steel material, and the elastic material membrane that is used to seal is a silica gel.Chip material is gold-plated glass, and all the other structures are with embodiment 1.
Under the effect of external pressure, silicon chip closely contacts with microarray, has just formed 100 independently cavitys of sealing like this between silicon chip and the groove.Each cavity has two fluid channel of one-in-and-one-out.Under the promotion of micro-ram pump, 100 kinds of protein solutions enter cavity and silicon chip surface contact reaction by fluid channel, thereby proteinaceous solid is fixed on the silicon chip surface.Re-use buffer solution for cleaning silicon chip surface, cavity and fluid channel, the protein that is not fixed on the silicon chip surface is discharged.Take off the stainless steel pipe, use pellosil to seal organic groove on glass, 100 grooves are together in series.Under the promotion of micro-ram pump, solution to be measured enters cavity and the proteins react that is fixed on the silicon chip surface from the import of plexiglass block side.Solution to be measured flows through 100 cavitys successively, discharges from outlet at last, uses buffer solution for cleaning then.The silicon chip that has reacted takes off detection from microarray.
Make second kind of device (shown in Fig. 4,5,7), comprising: fluid channel 5 and the microarray template that is complementary with it; Wherein the microarray template comprises: an area be the sheet rubber of 20mm * 20mm as elastic material block 3, be carved with the elastic material block 3 of 100 grooves 2 on its surface, each strip groove 2 degree of depth is that 0.01mm, sectional area are 0.1mm
2100 are divided into 10 rows and are arranged in regularly on the sheet rubber 1, and a row has 10 grooves 3.Facing up of elastic material block 3 fluted 2, a through hole 10 is opened at the two ends of each strip groove 2 respectively, and these through hole 4 internal diameters are 0.1mm, totally 200.The another side of block rubber 3 closely contacts with chip 1 surface; Fluid channel is produced on the pellosil 13, on pellosil 13, be shaped on wide 0.1mm, the chase 14 of dark 0.1mm is produced on through hole 10 positions of the corresponding microarray upper groove 2 of an end of its chase 14, and the another port 6 of chase 14 extends to pellosil 13 edges and extraneous UNICOM; Through hole 10 corresponding chases 14 of each groove; And the film of corresponding microarray pellosil 13 through holes on the pellosil 13 is got through pore-forming, on be with chase 14 pellosils 13 again, cover one deck plastic foil 16 again, and pellosil 13 and plastic foil 16 two membranes peripheries are bonded together, formed fluid channel 5 by chase 14, have wide 0.1mm between the fluid channel 5, the chase of dark 0.1mm interconnects it, at node location, the method for pushing by external force forms first, second switch 15,17.
Also comprising on the plexiglass block 4 as rigid solid soon 4 on the basis of embodiment 4, this solid pieces of material 4 is the plexiglass block of 25mm * 25mm; One area is the sheet rubber 3 of 20mm * 20mm, and this sheet rubber 3 coheres at 3 strip grooves 2 that are carved with degree of depth 0.01mm on sheet rubber 1 on the plexiglass block 4, and totally 100, each strip groove 2 sectional area is 0.1mm
2100 are divided into 10 rows and are arranged in regularly on the sheet rubber 3, and a row has 10 grooves 2.A through hole 10 is opened at the two ends of 100 grooves 2 on the elastic material sheet 3 of this substrate respectively, and these through hole 4 internal diameters are 0.1mm, totally 200.Fluid channel is produced on the pellosil 13, has wide 0.1mm on pellosil 13, dark 0.1mm chase 14 (shown in Fig. 4,6,7).
Under the effect of external pressure, silicon chip closely contacts with microarray, forms 100 cavitys.During application of sample, close the second switch II between fluid channel, each groove part has and independently passes in and out runner.Under the driving of ram pump, different protein solutions enters into cavity and silicon chip surface reaction, thereby fixes from the teeth outwards.Use buffer solution for cleaning then, remove and be not fixed on proteins on surfaces.Open the second switch 17 between fluid channel, close first switch 15 on the fluid channel, so just 100 cavitys are cascaded, only stay 6, one outlets of next import.Protein solution to be measured enters each cavity successively and is fixed on the proteins on surfaces reaction along import 6.After the reaction, use buffer solution for cleaning.Silicon chip takes off detection from device.
Claims (13)
1. method that is used for biomolecule chip micro sample-adding and reaction comprises that order is carried out as follows:
(1) base material with chip closely contacts with the microarray template, and compresses fluid channel by external force;
(2) the fine pipe of aglucon molecule by fluid channel is transported to the lip-deep selection area of chip base; By the time after the aglucon molecule is fixed on the chip base;
(3) chip base that is fixed with biomolecule that step (2) is prepared is washed with damping fluid, damping fluid is transported in the zones of different of chip surface by fluid channel; Wash the aglucon molecule that is not fixed on the chip surface;
(4) by a flexible sheet, the zone that is fixed with the aglucon molecule is together in series; Perhaps the switch that forms by the node on the extruding flexible sheet is together in series the zone that is fixed with the aglucon molecule;
(5) biological sample to be detected is transported in each unit on the chip surface that step (4) prepares by fluid channel again, carries out detection reaction immediately;
(6) take off step (5) preparation and the chip that is loaded with the detection reaction result that obtains, use chip detector to detect its reaction result.
2. the method that is used for biomolecule chip micro sample-adding and reaction as claimed in claim 1 is characterized in that: described chip base material comprises: the silicon chip of silicon chip or gold-plated film.
3. the method that is used for biomolecule chip micro sample-adding and reaction as claimed in claim 1 is characterized in that: described aglucon molecule is a protein, DNA, and its aglucon molecular conecentration is 0.001-1mg/ml.
4. the method that is used for biomolecule chip micro sample-adding and reaction as claimed in claim 1 is characterized in that the flow velocity of aglucon molecule in fluid channel is the 0.1-100 mul/min in the step (3).
5. the described isolated plant that is used for the method for biomolecule chip micro sample-adding and reaction of claim 1 comprises: fluid channel (5); It is characterized in that: also comprise the microarray template that is complementary with its fluid channel (5); This microarray template comprises: be carved with the elastic material sheet (3) of groove (2) on surface, its groove (2) is arranged by the array formula, and groove (2) two ends have first through hole (10); Also be carved with groove (2 ') on one surface of a rigid solid material block (4), the two ends of groove (2 ') have second through hole (11), and the groove one side that is carved with of elastic material sheet (3) is not with groove one side relative fixed together with rigid solid material block (4); Through hole on this rigid solid material block (4) and through hole on the elastic material sheet (3) stagger one corresponding and communicate, solid pieces of material (4) two sides have a testing liquid import (7) and a testing liquid outlet (8), and a switch is installed in the import; Chip (1) closely contact on elastic material sheet (3), the pipe that the plug-in mounting diameter is close in the through hole of solid pieces of material (4) (12), fluid channel is with the formation that is connected of the through hole on the microarray template by pipe (12).
6. by the described isolated plant that is used for the method for biomolecule chip micro sample-adding and reaction of claim 5; It is characterized in that: the elastic membrane (9) that also comprises a closed pockets (2); This elastic membrane (9) is covered on the solid pieces of material of taking off pipe (12) (4), open a hole in the elastic material block relative (3) side-walls with solution inlet port, as detecting liquid import (7), with the hole that solid pieces of material (4) side is opened as detecting taphole (8), perhaps have one as the hole of detecting taphole (8) and import (7) in the solid pieces of material relative (4) two sides with solution inlet port.
7. described isolated plant that is used for the method for biomolecule chip micro sample-adding and reaction of claim 1, comprising: fluid channel (5) is characterized in that: also comprise the microarray template that is complementary with it; Wherein the microarray template comprises: the elastic material sheet (3) that is carved with at least 2 grooves (2) on the surface, facing up of elastic material sheet (3) fluted (2), a through hole (10) is opened at the two ends of each groove (2) respectively, and the another side of elastic material sheet (3) closely contacts with chip (1) surface; Fluid channel comprises: a resilient material diaphragm (13), on resilient material diaphragm (13), be shaped on chase (14), and produce another port (6) and extraneous UNICOM on through hole (10) position of the corresponding microarray upper groove of an end of its chase (14) (2); The corresponding chase (14) of the through hole of each groove (10); And the film of resilient material diaphragm (13) being gone up corresponding microarray resilient material diaphragm (13) through hole is got through pore-forming, on be with chase (14) resilient material diaphragm (13) again, cover one deck elastic material membrane again, and the two membranes periphery is bonded together, formed fluid channel (5) by chase (14), fluid channel interconnects between (5), at node location, the method for pushing by external force forms first, second switch (15), (17).
8. by the described isolated plant that is used for the method for biomolecule chip micro sample-adding and reaction of claim 7; It is characterized in that: also comprise a rigid solid material block (4), this solid pieces of material (4) is fixed on the face of resilient material diaphragm (3) band groove (2), the through hole (10) that open at the two ends of elastic material membrane piece (3) groove (2) runs through rigid solid material block (4), and solid pieces of material (4) upper surface that resilient material diaphragm (16) does not have the elastic material membrane and the microarray of chase is bonded together.
9. by claim 5,6, the 7 or 8 described isolated plants that are used for the method for biomolecule chip micro sample-adding and reaction; It is characterized in that: described elastic material sheet (3) comprising: silica gel, rubber, plastics or other rubber-like material.
10. by claim 5,6, the 7 or 8 described devices that are used for directly preparing the biomolecule chip and carry out detection reaction immediately; It is characterized in that: described solid pieces of material (4) comprising: materials such as silica gel, rubber, plastics, metal, glass, its thickness 1mm is to 10mm.
11. by claim 5,6, the 7 or 8 described isolated plants that are used for the method for biomolecule chip micro sample-adding and reaction; It is characterized in that: described groove (2) or (2 ') are strip groove, and strip groove is 2-1000; Its strip groove area is from 0.01mm
2-1mm
2The degree of depth is from 10 μ m-1mm.
12. by claim 5 or the 7 described devices that are used for directly preparing the biomolecule chip and carry out detection reaction immediately; It is characterized in that: the internal diameter of described through hole (10) or (11) from 10 μ m to 1mm.
13. by the described device that is used for directly preparing the biomolecule chip and carries out detection reaction immediately of claim 7; It is characterized in that: the internal diameter of described chase (14) from 10 μ m to 1mm.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03102659 CN1249437C (en) | 2003-02-17 | 2003-02-17 | Method and apparatus for bio-molecular chip minute quantity sample application and reaction |
| PCT/CN2004/000074 WO2004072642A1 (en) | 2003-02-17 | 2004-01-20 | A device for application of micro-sample and reaction of a biochip and its method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03102659 CN1249437C (en) | 2003-02-17 | 2003-02-17 | Method and apparatus for bio-molecular chip minute quantity sample application and reaction |
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| Publication Number | Publication Date |
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| CN1523354A true CN1523354A (en) | 2004-08-25 |
| CN1249437C CN1249437C (en) | 2006-04-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03102659 Expired - Fee Related CN1249437C (en) | 2003-02-17 | 2003-02-17 | Method and apparatus for bio-molecular chip minute quantity sample application and reaction |
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| Country | Link |
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| CN (1) | CN1249437C (en) |
| WO (1) | WO2004072642A1 (en) |
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| CN101038290B (en) * | 2006-03-17 | 2011-05-11 | 中国科学院力学研究所 | Bilayer lipid membrane surface modified protein chip and its manufacturing method and use |
| CN102242053A (en) * | 2011-04-01 | 2011-11-16 | 沈越 | Biochip with polymer three-dimensional nanostructure |
| WO2012126294A1 (en) * | 2011-03-22 | 2012-09-27 | 博奥生物有限公司 | Biological chip hybridization system |
| CN103558135A (en) * | 2013-11-07 | 2014-02-05 | 西南石油大学 | Repeatedly-usable glass chip model |
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| CA2449193C (en) * | 2001-06-08 | 2010-04-20 | Centre National De La Recherche Scientifique | Method of manufacturing a microfluidic structure, in particular a biochip, and structure obtained by said method |
| DE10142789C1 (en) * | 2001-08-31 | 2003-05-28 | Advalytix Ag | Movement element for small amounts of liquid |
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2003
- 2003-02-17 CN CN 03102659 patent/CN1249437C/en not_active Expired - Fee Related
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2004
- 2004-01-20 WO PCT/CN2004/000074 patent/WO2004072642A1/en active Application Filing
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2004072642A1 (en) | 2004-08-26 |
| CN1249437C (en) | 2006-04-05 |
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